Stem cell expansion: control cell
environment during scale up with a
multiplate bioreactor.
NBV symposium
New initiatives in animal cell culture : Developments in
stem cell cultivation
November 22, 2012
Egloff Matthieu, Product Manager
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AGENDA
ATMI Lifesciences Introduction
Scaling up stem cell culture
Learning from our customers
Xpansion Multiplate Bioreactor
Importance of controlling cell culture environnement – A
case study : Promethera Biosciences
Enabling cell therapy manufactuing
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Artelis’
Mission
ATMI – Who
we are
ATMI LS mission is to be the source of Biopharm process efficiency by providing
comprehensive disposable technology solutions that maximize product yield and
integrity
Advance Technology Materials, Inc.
Founded in 1986; IPO in 1992
2011 Revenue $390 M
>700 Global employees
LifeSciences and Semiconductor
business focus
Largest Ultra Clean liner company in the
world with applications in semi, flat panel
& life sciences (>$110 M )
ATMI LifeSciences is a market leader in
ultra-clean films, critical fluid handling
and bioprocess solutions
3
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Integrity™ Line of Single-Use Technologies
Sterilizable Packaging (NewformTM)
Ultraclean
Sterilizable by autoclave, gamma or EtO
Custom films; custom sizes
INTEGRITY Liquid and Powder Vessels
Liquid Storage: BioProcess Vessels (BPVs)
Powder Storage: Anti-static Powder Transfer Vessels (PTVs)
Supporting accessories
INTEGRITY Mixers
Fully contained, sterile mixing and storage systems
Powder-liquid, powder-powder and liquid-liquid mixing
Scalable from 1L to 2000L
INTEGRITY Bioreactors
Fully-integrated NucleoTM or “open architecture” PadReactorTM
iCellisTM fixed bed bioreactor
XpansionTM stem cellbioreactor
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Artelis’
Mission
ATMI “problem
solving” approach
Applications & Development Center
ATMI has an approach combining both:
Product development and manufacturing
Process development and optimization
ATMI has an Applications & Development center:
Located on the Solvay Group R&T site (Brussels)
500m², 4 fully equipped clean rooms. BL2 and BL3 GMP-grade
suites, Class 10,000 (ISO7) environment
45 experienced team including:
•
Engineers – product design and development
•
Bioengineers & biologists with former experience in the
industry – cell culture process development
PD for New Bioreactor development
PD Services / Support implementation of our technologies
Tech transfer to your facility or to your CMO
5
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Learning from our customers
(back to 2008)
• Discussions with cell therapy companies (EU and USA)
• Autologous model high dose / 0,5-2,0 bns cells (MSCs)/batch
• ~10,000 patients / year, 3 sites – 3000 treatments per site
Manufacturing challenges
3000 treatments / years:
Issues
Patient safety

20 MLS10 are needed per batch
QA/QC challenge – Regulatory

200 aseptic operations per batch related to
process
Consistency
600.000 aseptic operations a year
~2.000 aseptic operations a day
Huge investment with current
•
No technologies available for large volume mgt
technologies
•
Very high level of CR classification & QA/QC
validation
Costs limitation
Need : Process development and bioreactor design for MSC’s expansion
6
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Learning from our customers
Design fixed by mid-2009
User Requirements & Specifications
our rationale
To ensure same microenvironment, we need to keep:
“We want a bioreactor
that allows:
•
•
To replace ~20 MLS10, in a
fully closed system, with a
controlled growth process
Future capacity increase
to > 150 cultures in
parallel, with a minimum
investment in equipment
BUT without any change in
the microenvironment of cell
growth, to minimize RA risks
Same material: polystyrene
Same type of geometry: plates
Same manufacturing: injection molding
Same surface treatment: electrostatic degradation
Where is space “lost” in standard stacked tray technologies:
Thickness of plates: 3mm
→ Reduce it to 1.0mm (challenge !)
Distance between plates: 15mm
→ Reduce it to 1.6mm
Now, we have a stack of plates, like in a MLS, with the same
ratio Surface area/Volume culture medium, whit gas
exchange means and whith:
Sensing, pH, pO2, T, biomass
Homogenization means
With inlets/outlets for gases/liquids
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Xpansion - Bioreactor design
XpansionTM bioreactor is made of :
A stacking of circular plates
Advanced aeration system
pH, pO2, T sensors
Means for biomass observation
Inlets/outlets for gases/liquids
Bottom pumping system
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Xpansion - Controller
The controller is a generic system installed on separate
skid for measurement & control
Several Features of Xpansion controller:
Graphical user interface with color display and touch screen
operation with security levels: Administrator, operator.
Integrated control loops for DO and pH
Calibration of pre-calibrated DO and pH sensors: Sensor patches
come precalibrated.
In-process DO and pH recalibration is possible
Agitation control (Speed range from 5 up to 250 rpm)
Temperature monitoring
Trend display for up to 4 process values (pH, DO, T, Q)
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Xpansion – regulation strategy
Gas exchange for ph / DO regulation
Stacking of plates creates a central
column by design
Central column incorporates a
silicon coil for gas transfer
Media homogeneization
Plates incorporate radial
slots/apertures
Bottom incorporates a pumping
system
All together allows internal
circulation and aeration of culture
medium
10
Youtube / Xpansion bioreactor
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Xpansion bioreactors : compact design
4m
0,8m
Xpansion
MPB-10
Xpansion
MPB-50
Xpansion
MPB-100
Xpansion
MPB-200
10
50
100
200
Growth Surface
Area
6120 cm²
30600 cm²
61200 cm²
122400 cm²
Media working
volume
1,6L
5,6L
11,2L
21,9L
29 cm (11.4 '')
47 cm (18.5 '')
55 cm (21.6 '')
81 cm (31.9 '')
1
5
10
20
# of plates
Size
Multitray 10stack equivalent
Xpansion 200 plates
bioreactor offer the
same surface as 20
multitray stack 10 plates
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Importance of controlling cell culture
environnement
Enabling control of cell culture parameters increase process scale up
efficiency
-

Same surface material and geometry as multitray stack
Control of the stem cell niche microenvironnement
Preserve stem cell integrity & facilitate transfert from multitray stack

Regulation of pH and DO
Control of the physiochemical environnement
Increase process reproductibility & robustness

Automated cell counting with holographic microscope
Control of the cell density and morphology
Mitigate quality risk & harvest time
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Case study – Promethera Biosciences
Promethera Biosciences®
Belgium Biotech company
Cell therapy product to treat children suffering from serious congenital
metabolic liver diseases.
HepaStem: human heterologous adult liver progenitor cells (ADHLSCs)
isolated from healthy human livers.
Phase I/II clinical trials treatment of Crigler Najjar (CN) and Urea cycle
disorders (UCD) in a pediatric setting
Using Corning CellBind treated CellStacks
Scale up allogeneics production to support clinical phase and early
commercialization
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Control of the surface material and
geometry : 2D surface
Similar cell growth from multitray stack to Xpansion 180 plates
% of cell density at harvest in Xpansion bioreactors VS Cellstack 2 control
140
120
100
80
Xpansion
60
CS2 CTRL
40
20
0
Xpansion 10
Xpansion 50
Xpansion 180
Viability at harvest
97%
98,7%
99%
FACS
Pass
Pass
In progress
ICC
Pass
Pass
In progress
Morphology
Pass
Pass
In progress
Activity of CYP3A4
Pass
Pass
In progress
Urea secretion
Pass
Pass
In progress
Bilirubin conjugation
Pass
Pass
In progress
QC test
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Regulation of pH and DO
Regulation of pH & DO enable to increase process reproductibility
Xpansion 10 :pH, DO, T trends
Xpansion 180 : pH, DO, T trends
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Cell density monitoring
Automatic cell counting support cell density monitoring and secure
harvest time
Observation of cells in the 3 scale of Xpansion, using
the holographic microscope - day of harvest
Automatic cell counting of cells in
Xpansion 10, using the holographic
microscope
Cell confluency : 50 %
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Xpansion - Cell Cultures performance
MSCs, ADSCs, Hepatocyte progenitors grow in XP technology:
Confluence, morphology and cell growth comparable to CS
Cell harvested: 90% viability (after centrifugation)
Homogeneity inter/intra plates
QC PASS
Xpansion was able to maintain pH, DO, T and homogeneity
Vero, MRC-5, A549 derived cells grow in XP technology:
Confluence and cell growth comparable to Tflasks
Homogeneity inter/intra plates
Xpansion was able to maintain pH, DO, T and homogeneity
Microscopy observation:
Cells density and morphology on the 10 top plates can be monitored
Scaling-up completed:
In house and at customer sites
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Learning from our customers
Bioreactor design
Xpansion design achieves URS
User Requirements & Specifications
“We want a bioreactor
that allows:
•
To replace 20 MLS10, in a
fully closed system, with a
controlled growth process
•
Future capacity increase
to > 150 cultures in
parallel, with a minimum
investment in equipment
Up to 120000cm² in one container,
equivalent to 20MLS
… without changes in the
microenvironment of cells
… leads to similar behavior vs
reference processes for several
biological materials, as proven by
our customers
... That can be controlled, both
physical parameters and cell growth
by µscopy
BUT without any change in
the microenvironment of cell
growth, to minimize RA risks
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Value proposition
By ensuring product safety and consistency, XpansionTM is an enabling
technology for stem cells manufacturing:
Xpansion allows the customer to benefit from all the advantages of
a bioreactor, BUT without any change in the µenvironment of cells,
which is UNIQUE
Xpansion allows mitigation of RA risks
Dramatic reduction of # of
operation
Process efficiency
Closed sytem
No change in the structure
of cell growth
Process safety
Process quality
consistency
Dramatic increase of safety
Significant reduction of regulatory risk
scalability
Mitigate risk
Incresa succes rate during clinical
phase
Process industrialization becomes possible
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Integrated Solution for Cell Therapies
Closed system from pre-culture to final filling to avoid aseptic operations
Single use
Primary culture kit
Harvest
ATMI primary
cell culture kit
Single use
Cell expansion kit
Single use
Centrifugation Kit
ATMI Xpansion
bioreactor
Detachment
Washing
1
3
Washing
2
Harvest
Cryopreservation
solution
6
7
5
Media
Bioreactor
harvest
Media
ATMI
Centrifugation
technology
Final
container
for
treatment
Waste
Rinsing
4
Waste
ATMI Integrity™ Bag
Tubing
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ATMI Lifesciences and cell therapies
manufacturing – future developments
Support large volume manufacturing ( High volume
commercial production )
Padreactor™
Allogeneic CT – large dose: Need to increase volumetric
productivity
ATMI developments:
Stay in 2D –move to micro-carriers, with very low shear
systems : ATMI padreactor
iCELLis™
Move to 3D – packed bed system can be a good way
to scale-up :ATMI iCELLis based technology
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2009 ATMI,
Inc.
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